Fungal infections are increasing as a result of AIDS, transplantation, chemotherapy, steroids and antibiotics, and invasive procedures and medical devices. Antifungal agents were limited to amphotericin B, flucytosine, and azoles, but now the candins, second-generation azoles, and lipid based amphotericin formulations have expanded the antifungal drug armamentarium. Yet with difficulties in delivering parenteral agents, consistent efficacy, need for rapid, short courses of therapy, and emerging drug resistance, therapeutic advances remain to be achieved. Our research focuses on signaling cascades as targets for antifungal drugs. Studies are proposed on Candida albicans, the most common human fungal pathogen that remains a major mucosal pathogen in AIDS patients who fail or do not receive HAART, and Cryptococcus neoformans, the leading cause of fungal meningitis in the world due to the AIDS epidemic. Our studies have defined the mechanisms of action and targets for the antifungal immunosuppressants cyclosporin A, FK506, and rapamycin. Fungal homologs of calcineurin, cyclophilin, FKBP12, and Tor1 were identified, providing insight into biological roles and as conserved drug targets. Nonimmunosuppressive analogs that retain antifungal activity were identified. Synergistic fungicidal drug interactions were demonstrated and mechanisms of action elucidated. Calcineurin inhibition by cyclosporin A or FK506 is potently synergistic with azoles against C. albicans and of therapeutic benefit in animal models. Recent studies implicate calcineurin as an Hsp90 client protein, and Hsp90 mutations or inhibitors are also synergistic with azoles. Here we propose to define Tor, calcineurin, and FKBP12 pathways as targets for therapy. First, we will characterize Tor cascade elements and functions and target this pathway with rapamycin and less immunosuppressive rapamycin analogs (rapalogs). Second, we will elucidate relationships between Hsp90 and calcineurin and their inhibitors that render azoles fungicidal and target this pathway with novel Hsp90 inhibitors, azoles, and calcineurin inhibitors. Third, we will focus on FKBP12 control of an amino biosynthetic cascade targeted by known antifungal agents and define synergistic antifungal drug combinations. Finally, drugs, analogs, and combinations will be tested in animal models of cryptococcosis and candidiasis. Our assembled team of collaborators in natural products, medicinal chemistry, enzymology, structural biology, and animal models complements our expertise in signaling and target identification. The goal is to harness signaling cascades to develop novel antifungal therapies.